An early response to tissue hypoxia is induction of hypoxia inducible factor (HIF), a basic helix-loop-helix transcriptional activator that mediates changes in gene expression in response to changes in cellular oxygen concentration. Hypoxia, a state of reduced oxygen, can occur when the lungs are compromised or blood flow is reduced. Ischemia, reduction in blood flow, can be caused by the obstruction of an artery or vein by a blood clot (thrombus) or by any foreign circulating matter (embolus), or by a vascular disorder such as atherosclerosis.
Modern drug discovery has been accelerated by the use of cell-based assays. Currently, these assays are utilized for lead identification, drug dose optimization as well as target validation. The use of small-scale yet high-throughput techniques makes these cell-based assays more efficient and cheap for drug screening. These assays are preferred over other in-vitro methods since they give direct cellular functional responses to the drug target of interest. This allows for clearer understanding of physiological and pharmacological responses to the target.
Currently, demand for assays to quantitate HIF activities is tremendous. This is due to the fact that besides being involved in various cellular regulations from proliferation, apoptosis to cancer development, HIF has also been shown to be involved in stem cell renewal and differentiation (Keith and Simon, 2007). The boost of stem cell research in the last decade has raised the need for a more robust cell-based assay system to measure HIF activities particularly in HIF-related drug screening. Unfortunately, the systems that are currently available are very inefficient, costly, time consuming and most importantly provide limited sensitivity. The choice of cell lines is also restrictive.
Development of cell-based HIF assay systems have been previously reported (Woldemichael et al., 2006; Ji et al., 2008). However, up to now only two HIF assay cell lines are commercially available. One is from Invitrogen, named ‘CellSensor® HRE-bla ME-180 Cell Line’ (cat# K1644). The other is from Panomics, under the name ‘Stable Cell Line: NIH3T3/HIF-luc’ (cat # RC0017). SABiosciences, a Qiagen company, also sells HIF assay kits, however, their kits are in the form of ready-to-transduce lentiviral particles or ready-to-transfect plasmids. Recently, the Emory University and The University of Rochester have also reported developments of reporter cell lines for HIF activity assay in their technology transfer catalogs. The signal ratio of hypoxia versus normoxia for the commercially-available CellSensor HRE-bla (Invitrogen) is just 13 fold. While another system developed by Li et al., (2008), only gave 14-fold difference. In the case of an unfavourable signal to noise ratio in cell based assays, such low inducibilities might render the application of these systems in a high throughput format for drug screening impossible.
In view of the above known reporter constructs of HIF activity it is an object of the present invention to provide an HIF responsive system which shows higher inducibility and specificity to HIF activation, specifically in response to hypoxia. Therefore, a further object of the present invention is to provide novel means for drug screening of modulators of HIF response, for example systems that are suitable to be applied in a high throughput format.
The above problem is solved in a first aspect by a gene construct which has a reporter gene integrated under the control of a hypoxia inducible factor (HIF) responsive promoter, wherein the HIF responsive promoter comprises at least one hypoxia-response element (HRE) derived from an erythropoietin (EPO) gene.
A gene construct according to the present invention shall refer to a nucleic acid molecule composed of several functional elements to allow for the expression of a reporter gene. The nucleic acid used in the context of the present invention can be single or double stranded; can be DNA or other nucleic acid molecules known in the art.
A promoter is a nucleic acid sequence that directs the transcription of a structural gene. Typically a promoter is located in the 5′ region of a gene, proximal to the transcriptional start site of a structural gene. The promoter of the invention comprises HRE element(s). Additionally, the promoter may include upstream elements. Such elements include UARs and optionally, other nucleic acid sequences that affect transcription of a structural gene such as a synthetic upstream element or an enhancer.
A core promoter or minimal promoter contains the essential nucleotide sequences for expression of the operable linked coding sequence, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
In one embodiment of the present invention, the promoter of the gene construct comprises at least 4 HREs. The HREs in the construct are located preferably in a tandem repeat, most preferably wherein the promoter is upstream of the reporter gene. Upstream of the reporter gene shall mean that the promoter sequence is located 5′ of the start codon of the reporter gene, even more preferably wherein the HRE of the invention are located 5′ of a TATA box upstream of the start codon of the reporter gene. In a preferred embodiment the construct of the present invention is depicted schematically in FIG. 2.
Another preferred embodiment of the present invention relates to a construct wherein the HRE is derived from human the EPO promoter region.
In the context of the present invention any reporter gene known to the person of skill in the art may be used and operably linked to the above described HRE element. Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue light, the enzyme luciferase, which catalyzes a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed. A common reporter in bacteria is the E. coli lacZ gene, which encodes the protein beta-galactosidase. This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal. An example of a selectable-marker which is also a reporter in bacteria is the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol. Similar markers are known to work for vertebrate cell lines. Most preferred in the context of the present invention is the use of a luciferase, most preferably wherein the reporter gene is a firefly luciferase.
In another preferred embodiment the present invention relates to a gene construct comprising the exact 4 times the sequence of the human EPO HRE upstream of a TATA-box and the reporter gene.
The above object of the present invention is solved in another aspect by a vector comprising the gene construct or nucleic acid sequence as described herein. A vector within the meaning of the present invention is a nucleic acid that is capable of being introduced into a cell. It is preferred that the proteins encoded by the introduced nucleic acids are expressed within the cell upon introduction of the vector. In a preferred embodiment, the vector of the present invention comprises recombinant vectors, plasmids, phagemids, phages, cosmids, viruses, in particular but not limited to virus-derived amplicon vectors.
The object of the present invention is furthermore solved by a cell line that comprises the gene construct or the vector, as described in the various embodiments of the present invention. The gene construct or vector may be transiently or stably transfected (or transduced) into said cells. It is preferred that the cells used for transfection are vertebrate, most preferably mammalian or human cells.
In one embodiment it is preferred that the cell line of the invention comprises a stably integrated gene construct and/or vector of the invention.
In a most preferred embodiment the cell line is an osteosarcoma cell line, preferably wherein the cell line is Saos-2.
In the present invention, the inventors used the human osteosarcoma cell line, Saos-2, while others have used the ME-180 cervical carcinoma cell line (Invitrogen) and the NIH-3T3 mouse embryo fibroblast cell line (Panomics). A rat glioma cell line was also used (Research Tools catalog, Emory University). In the present study several human cancer cell lines were screened, including breast adenocarcinoma MCF7, cervical carcinoma HeLa, renal carcinoma 786-O and osteosarcoma Saos-2 cell lines. Luciferase signals that were produced by our reporter construct upon hypoxia induction were highest in the Saos-2 cells. Based on these novel findings, the development of a stable hypoxia reporter construct cell line, using the Saos-2, was conducted.
The problem posed is furthermore solved by a method for assaying HIF response in a cell, comprising the steps of:                a. introducing a gene construct according to any one of claims 2 and 4 into said cell,        b. incubating said cell in hypoxic conditions,        c. optionally, incubating a control cell in normoxic conditions, and        d. ensuring the reporter gene expression corresponds to the HIF response in said cell.        
In this aspect it is in another embodiment preferred that said cell is an osteosarcoma cell.
Furthermore, embodiments of the invention relate to the above method, wherein the gene construct is transduced or transfected into said cell, preferably wherein said gene construct is stably transduced or transfected into said cells.
In the context of the present invention, the term “hypoxia” shall refer to an oxygen (O2) concentration in the atmosphere that is between 0 and 5% O2, preferably between 0.01 and 1% O2, more preferably between 0.01 and 0.3% O2. In a most preferred embodiment hypoxia refers to a concentration of oxygen of about 0.3% O2.
On the other hand, in the context of the present invention the term “normoxia” shall denote a concentration of oxygen of between 5 and 21% O2, preferably between 10 and 21% O2, more preferably between 10 and 20% O2. In a most preferred embodiment the “normoxia” refers to a normal atmospheric oxygen concentration.
The problem of the present invention is furthermore solved by a screening method for identifying modulators of HIF response comprising, performing the above method of the invention, and contacting said cells with a compound which is suspected to be a modulator of HIF signaling, wherein an increase in reporter gene expression compared to an untreated control indicates that the compound is an agonist of HIF response, and wherein a decrease of reporter gene expression compared to an untreated control indicates that the compound is an antagonist of HIF response.
Suitable test compounds for use in the context of the present invention are small molecules comprised in compound libraries. Alternatively, proteins or peptides can be used.
Also nucleic acid molecules can be used as candidate compounds for the herein described, screening methods. Of these, inhibitory RNAs are of particular interest. This may be antagomirs, siRNAs, dsRNAs or antisense RNAs. Inhibitors RNA molecules are commercially available in libraries suitable for a screening approach in high throughput format.
Furthermore, the present invention provides a compound that modulates hypoxia response obtained by the above screening method.
In another aspect, the present invention relates to a kit for testing HIF response in a cell, comprising a gene construct, or a vector, or a cell according to any of the herein described embodiments of the invention.
Optionally the kit of the invention may include manual instructions how to perform the methods of the present invention and/or buffers and/or solutions for performing a HIF response test.
Furthermore, the invention relates to the use of the inventive material as shown herein, specifically the inventive gene construct, the vector, the cell or the kit, in a screening method for modulators of HIF mediated response or hypoxia induced response.
While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the invention within the principles and scope of the broadest interpretations and equivalent configurations thereof.